US4400316A - C-Terminal fragment of human chorionic gonadotropin - Google Patents

C-Terminal fragment of human chorionic gonadotropin Download PDF

Info

Publication number
US4400316A
US4400316A US06/320,699 US32069981A US4400316A US 4400316 A US4400316 A US 4400316A US 32069981 A US32069981 A US 32069981A US 4400316 A US4400316 A US 4400316A
Authority
US
United States
Prior art keywords
pro
added
ser
vacuo
sub
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US06/320,699
Other languages
English (en)
Inventor
Shigeo Katsuragi
Kaoru Morita
Sadami Kobari
Toshiharu Noda
Nobuaki Nakagawa
Susumu Watanabe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toyo Jozo KK
Original Assignee
Toyo Jozo KK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toyo Jozo KK filed Critical Toyo Jozo KK
Assigned to TOYO JOZO KABUSHIKI KAISHA, A CORP. OF JAPAN reassignment TOYO JOZO KABUSHIKI KAISHA, A CORP. OF JAPAN ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: KATSURAGI, SHIGEO, KOBARI, SADAMI, MORITA, KAORU, NAKAGAWA, NOBUAKI, NODA, TOSHIHARU, WATANABE, SUSUMU
Application granted granted Critical
Publication of US4400316A publication Critical patent/US4400316A/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C07K5/06069—Ser-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C07K5/06095—Arg-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/11—Gonadotropin; related peptides

Definitions

  • R 2 H or R 4 -Asp-His-Pro-Leu-Thr group
  • R 4 H-R 5 -Gly-Gly-Pro-Lys group
  • R 5 Cys or Tyr group
  • R 3 Cys or S-acetamidemethyl-Cys group
  • HCG is a glycoprotein hormone secreted from placenta at pregnancy, and has an important role for maintaining pregnancy. Quantitative or qualitative analysis of HCG can be used for the diagnosis of pregnancy, ectopic pregnancy or choriocarcinoma.
  • LH luteinizing hormone
  • FSH follicle-stimulating hormone
  • CG chorionic gonadotropin
  • the ⁇ -chain is specific for each gonadotropin, but the ⁇ -chains of LH and CG are quite similar. However, the amino acid sequence adjacent the C-terminal of the ⁇ -chain of CG can be differentiated; therefore, an accurate and reliable CG assaying system can be provided without confusion with the other gonadotropic hormones, especially LH.
  • the present invention makes use of this concept, and the antibody obtained from the antigen consisting of the novel peptide of formula [I] has immune crossing activity.
  • the peptide [I] is thus useful for the preparation of an antibody for assaying HCG or a labelling reagent.
  • An amino acid and/or lower peptide is reacted by condensation in the order of the amino acid sequence of formula [I], and the protective group for the reactive group is released at the final stage of the reaction.
  • the condensation reaction can be carried out by conventional peptide synthesis by repeating the attaching and removal of the protective groups and condensation.
  • the protective groups for the synthesis of the starting materials or intermediates are conventional protective groups for peptide synthesis and are easily removable by hydrolysis, acid decomposition, reduction, aminolysis or hydrazinolysis.
  • the amino group may be protected conventionally by an acyl group such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, p-toluenesulfonyl, o-nitrophenylsulfonyl or 2,4-dinitrophenylsulfonyl group; an aralkyl group such as benzyl, diphenylmethyl or triphenylmethyl (these groups may optionally be substituted with a lower alkoxy group such as o-methoxy or p-methoxy); a benzyloxycarbonyl group such as benzyloxycarbonyl, o-bromobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-phenylazobenzyloxycarbon
  • the carboxyl group can be protected by amide formation, hydrazide formation or esterification.
  • the amide group is substituted with a 3,4-dimethoxybenzyl or bis-(p-methoxy-phenyl)-methyl group.
  • the hydrazide group is substituted with a benzyloxycarbonyl, trichloroethyloxycarbonyl, trifluoroacetyl, t-butoxycarbonyl, trityl or 2-p-diphenyl-isopropoxycarbonyl group.
  • the ester group is substituted with an alkanol such as methanol, ethanol, t-butanol or cyanomethylalcohol; an aralkanol such as benzylalcohol, p-bromobenzylalcohol, p-chlorobenzylalcohol, p-methoxybenzylalcohol, p-nitrobenzylalcohol, 2,4,6-trimethylbenzylalcohol, benzhydrylalcohol, benzoylmethylalcohol, p-bromobenzoylmethylalcohol or p-chlorobenzoylmethylalcohol; a phenol such as 2,4,6-trichlorophenyl, 2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p-cyanophenol or p-methanesulfonylphenol; or a thiophenol such as thiophenol, thiocre
  • the hydroxy group is serine, threonine or tyrosine may optionally be protected by esterification or etherification.
  • a group protected by esterification is, for example, a lower alkanoyl group such as an acetyl group; an aroyl group such as a benzoyl group; or a group derived from carbonyl such as benzyloxycarbonyl or ethyloxycarbonyl.
  • a group protected by etherification is, for example, a benzyl, tetrahydropyranyl or t-butyl group.
  • Protection of the hydroxy group can be effected by a 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl or 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group. However it is not always necessary to protect these hydroxy groups.
  • the amino group in the guanidino group in arginine can be protected by a nitro, tosyl or benzyloxycarbonyl group. However it is not always necessary to protect the guanidino group.
  • the imino group in histidine can be protected by a benzyl, trityl, benzyloxycarbonyl, tosyl, adamantyloxycarbonyl, 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl or 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, although the imino group does not always require to be protected.
  • the mercapto group is cysteine can be protected by a benzyl, p-methoxybenzyl, p-nitrobenzyl, trityl, benzylthiomethyl, ethylcarbamoyl or acetamidemethyl group.
  • the peptide [I] is synthesized by the condensation of amino acids of lower peptides. For example, an amino acid or peptide having a protected ⁇ -amino group and an activated terminal carboxyl group is reacted with an amino acid or peptide having a free ⁇ -amino group and protected terminal carboxyl group. On the other hand, an amino acid or peptide having an activated ⁇ -amino group and protected terminal carboxyl group is reacted with amino acid or peptide having a free terminal carboxyl group and a protected ⁇ -amino group.
  • the carboxyl group can be activated by, for example, an acid azide, acid anhydride, acid imidazolide or active ester, such as by converting to cyanomethyl ester, thiophenylester, p-nitrophenylester, p-nitrothiophenylester, p-methanesulfonylphenylester, thiodylester, 2,4-dinitrophenylester, 2,4,5-trichlorophenylester, 2,4,6-trichlorophenylester, pentachlorophenylester, N-hydroxysuccinimide ester, N-hydroxyphthalimido ester, 8-hydroxyquinoline ester or N-hydroxypiperidine ester, carbodiimide, N,N'-carbonyldiimidazol or an isoxazolium salt such as Woodward reagent.
  • active ester such as by converting to cyanomethyl ester, thiophenylester, p-nitrophenylester
  • the preferred condensation reactions are the carbodiimide, azide, active ester and acid anhydride methods.
  • racemization should carefully be avoided, and the preferred methods are the azide, active ester method, Wunsch method [Z. Naturforsch., 216, 426 (1966)] or Geiger method [Chem. Ber., 103, 788 (1970)], especially using N-ethyl-N'-3-dimethylaminopropyl-carbodiimide (WSCI) as a condensation agent.
  • WSCI N-ethyl-N'-3-dimethylaminopropyl-carbodiimide
  • the process of the present invention is preceded by a condensation reaction in the amino acid sequence of the formula [I], and it is preferable to synthesize from the C-terminal.
  • the protected HCG [127-145] is preferably synthesized by a modified Geiger method using WSCI with condensation of the C-terminal fragment 132-145 and the N-terminal fragment 127-131.
  • the C-terminal fragment 132-145 is preferably synthesized by condensation of fragment 132-137 and fragment 138-144 by a modified Geiger method using WSCI.
  • the N-terminal fragment 127-131 is preferably synthesized by condensation of the fragment 127-129 and fragment 130-131 by a modified Geiger method using WSCI.
  • Protected HCG [118-145] is preferably synthesized by condensation of the C-terminal fragment 127-145, i.e. protected HCG [127-145] and the N-terminal fragment 118-126 by a modified Geiger method using WSCI.
  • the C-terminal fragment 118-126 is preferably synthesized by condensation of fragment 118-121 and fragment 122-126 by the azide method.
  • Protected HCG [105-145] is preferably synthesized by condensation of the C-terminal fragment 112-145 and the N-terminal fragment 105-111 by a modified Geiger method using WSCI.
  • the C-terminal fragment 112-145 is preferably synthesized by sequential condensation of protected HCG [118-145] and fragment 116-117, fragment 113-155 and the 112th amino acid by the active ester method.
  • the N-terminal fragment 105-111 is preferably condensed by the azide method from fragment 110-111 and fragment 105-109.
  • Protected HCG [100-145] is preferably condensed by the active ester method with the C-terminal fragment 112-145 and the N-terminal fragment 100-111.
  • the N-terminal fragment 100-111 is preferably condensed from the fragment 100-104 and the fragment 105-111 by the azide method.
  • Protected [Tyr 100 ]-HCG [100-145] is preferably synthesized by condensation of the C-terminal fragment 112-145 and the N-terminal fragment 100-111 by the active ester method.
  • the N-terminal fragment 100-111 is preferably condensed from the fragment 100-104 and the fragment 105-111 by the azide method.
  • the C-terminal carboxyl group need not always be protected.
  • the carboxyl group can be protected by esterification such as by formation of the methyl, ethyl or benzyl ester.
  • the ester group such as a methyl ester can be removed with dilute sodium hydroxide solution or by conversion of the hydrazide, and the benzyl ester group can be removed with anhydrous hydrogen fluoride or by catalytic hydrogenation.
  • the ⁇ -amino group of the peptide is protected by a conventional protective group, such as a benzyloxycarbonyl, t-butoxycarbonyl or t-amyloxycarbonyl group.
  • a conventional protective group such as a benzyloxycarbonyl, t-butoxycarbonyl or t-amyloxycarbonyl group.
  • the benzyloxycarbonyl group is removed by catalytic hydrogenation and the t-butoxycarbonyl and t-amyloxycarbonyl groups are removed by trifluoroacetic acid.
  • the preferred protective groups are: the hydroxyl groups of serine and threonine by the benzyl groups; the hydroxyl group of tyrosine by a 2,6-dichlorobenzyl group; ⁇ -amino group of lysine by an o-chlorobenzyloxycarbonyl group; the amino group in the guadinino group of arginine by a tosyl group; and the mercapto group of cysteine by a p-methoxybenzyl group.
  • These protective groups can be removed by anhydrous hydrogen fluoride.
  • An acetamide methyl group can be used as a protective group for the mercapto group of cysteine. Since this group is not removed by anhydrous hydrogen fluoride, it can be removed by mercuric acetate at pH 4 at the time of removal of the other groups.
  • protected HCG [127-145], protected HCG [118-145], protected HCG [105-145], protected HCG [100-145] and protected [Tyr 100 ]-HCG [100-145] are obtained.
  • Their protective groups are preferably split by acid decomposition such as one-step removal with anhydrous hydrogen fluoride to obtain the corresponding compound of the formula [I].
  • the mercapto group of the 110th and/or 100th cysteine When the mercapto group of the 110th and/or 100th cysteine is protected by an acetamide methyl group, it can be removed with mercuric acetate at pH 4 after removal of the other protective groups with anhydrous hydrogen fluoride.
  • the above compound [I] can be purified by known purification methods for peptides. For example, it can be purified by column chromatography using Sephadex LH-20 (trade name), Sephadex G-50 (trade name), Dowex 1 (trade name) and carboxy methyl cellulose.
  • the peptide [I] can be obtained in the form of the base or its salt, preferably its salt with an organic acid such as acetic acid.
  • HCG can be assayed by immune reaction using antibodies obtained from peptide [I] as an antigen, or by using peptide [I] itself as an antigen, by any of the known techniques of enzyme immuno assay, radio immuno assay, a hemagglutination inhibition reaction, or a hemagglutination reaction or latex fixation test. Examples are illustrated hereinbelow.
  • the thus-obtained antibodies are designated as Lot A-2, Lot B-3, Lot C-2, Lot D-1 and Lot E-1.
  • WSCI 200 mg was added to a solution of BSA (bovine serum albumin) (30 mg) and HCG [127-145] (60 mg) dissolved in 0.1 M phosphate buffer (pH 8.0, 1 ml) and stirred at room temperature for 30 minutes.
  • the reaction mixture was passed through a column of Sephadex G-50 (1.5 ⁇ 50 cm) with distilled water for gel-filtration.
  • the fraction containing BSA-HCG [127-145] was collected and lyophilized to obtain BSA-HCG [127-145] (34 mg).
  • the product (1 mg) dissolved in physiological saline (2 ml) was injected into guinea pigs as described in Example 1 and the blood was collected.
  • the antibody obtained was designated Lot F-1.
  • BSA 60 mg
  • 2,2'-dithiobenzothiazyl propionic acid 0.2 mg
  • 0.1 M phosphate buffer pH 7.5, 2 ml
  • a solution of 2,2'-dithiobenzothiazyl propionic acid succinimide ester (6 mg) in DMF (0.3 ml) was added thereto and the mixture was stirred at 0° C. for one hour.
  • the reaction mixture was adjusted to pH 5 by adding 1 N HCl and was passed through a column of Sephadex G-50 (1.5 ⁇ 50 cm) using 0.1 M phosphate buffer (pH 7.5) for gel filtration. The fractions at 27-35 ml were collected.
  • the product (1 mg) dissolved in physiological saline (2 ml) was injected in guinea pigs as in Example 1 and the blood collected.
  • the thus-obtained antibody is designated as Lot G-1. [Diagnosis of pregnant urine by the hemagglutination inhibition test].
  • Diagnosis of pregnant urine was performed by the hemagglutination inhibition test according to the method described in Acta Endocrinol., 35, 261 (1960).
  • Carrier Silica-gel G.
  • Carrier Merck cellulose.
  • THF 300 ml was added to BOC-Leu-OH.H 2 O (149.59 g, 0.6 M). THF (150 ml) and DMF (100 ml) were added thereto to prepare a solution. Then H-Pro-OBzl (152.28 g, 0.63 M) was added thereto, and WSCI (120.8 ml, 0.66 M) was added dropwise at -10° C., and further DMF (100 ml) was added; then the mixture was stirred at room temperature overnight.
  • TFA 300 ml was added to the substance [1] (192 mM) dissolved in methylene chloride (100 ml), and the mixture was stirred at room temperature for 30 min. TFA was distilled off in vacuo. Hexane was added thereto, and distilled off in vacuo. The oily residue dissolved in THF (200 ml) was neutralized with NMM (56.1 ml) to pH 7 with ice cooling. BOC-Ile-OH.H 2 O (38.33 g, 159.5 mM) and HOBT (21.55 g, 159.5 mM) were added thereto.
  • Substance [2] (76.98 g, 145 mM) was dissolved in n-butanol (20 ml) and ethanol (300 ml). 5% Pd/C (15 g) was added thereto and catalytic hydrogenation was allowed to proceed for 3 hours. After removing the catalyst, the mother liquor was concentrated in vacuo. Diethyl ether (300 ml) was added to the residue, which was then extracted successively with 500 ml and 200 ml of 5% aqueous sodium bicarbonate. The extract was adjusted to pH 4 by adding 1 N HCl with ice cooling to precipitate the product continuously, which was extracted with ethyl acetate and washed twice with water.
  • TFA 150 ml was added to BOC-Gln-OBzl (36.41 g, 108 mM) dissolved in methylene chloride (100 ml) and the mixture was stirred at room temperature for 30 minutes.
  • the TFA was distilled off in vacuo and the residue was dissolved in THF (100 ml) which was adjusted to pH 7 by adding FNMM (40 ml) while cooling with a freezing mixture.
  • Substance [5] (52.78 g, 106.29 mM) was dissolved in methanol (300 ml). 1 N NaOH (150 ml, 1.4 molar excess) was added dropwise during 20 minutes with ice cooling and then the mixture was stirred at room temperature. 1 N HCl (43.71 ml, 0.4 molar excess) was added with ice cooling to adjust to pH 7 and methanol was distilled off in vacuo. The aqueous layer was washed with ether (150 ml) and the pH of the aqueous layer was adjusted to pH 3 by adding 1 N HCl (110 ml). The solution was extracted twice with ethyl acetate (300 ml and 150 ml).
  • the extract was dried by adding anhydrous sodium sulfate, then concentrated in vacuo. Ether and hexane were added to the residue to obtain the precipitate. The same operation was repeated and the precipitate was dissolved in ethyl acetate, then dried in vacuo to obtain the foaming solid substance [6] (32.27 g, yield: 77.75%).
  • Amino acid analysis [1.330 mg/HCl 0.3 ml, acetic acid 0.3 ml, anisole 0.1 ml, 105° C., 24 hours]: Thr 0.84 (1), Pro 2, Ile 0.97 (1), Leu 1.00 (1), Gln 0.98 (1).
  • BOC-Asp(OBzl)-OH 29.77 g, 87.72 mM
  • HOBT 11.85 g, 87.72 mM
  • WSCI 16.05 ml, 87.72 mM
  • the solvent was distilled off and water was added to the residue, then the precipitate formed was separated by decantation with ice cooling.
  • the solvent was distilled off in vacuo, ethyl acetate (500 ml) was added, and the mixture was washed twice with 5% sodium bicarbonate, saturated sodium chloride solution, twice with 1 N HCl, twice with saturated sodium chloride solution and water, in that order.
  • the ethyl acetate layer was dried with anhydrous sodium sulfate, and the mixture was concentrated in vacuo to obtain an oily material (67 g) which was dissolved in benzene (100 ml).
  • the solution was charged on a column (7 ⁇ 33 cm) of silica gel (500 g) packed with benzene, through which further benzene (300 ml) was passed.
  • TFA (220 ml) was added to the substance [12] (68.72 g, 120 mM) dissolved in methylene chloride (100 ml) and the mixture was stirred at room temperature for 20 minutes.
  • TFA was distilled off at 0° C. in vacuo, hexane was added thereto and the mixture was dried in vacuo.
  • the residue was dried over potassium hydroxide in a desiccator, dissolved in DMF (200 ml) and adjusted to pH 6 by adding NMM (59.4 ml) at -10° C.
  • the precipitate was extracted twice with ethyl acetate (500 ml). The extract was washed with 5% sodium bicarbonate solution, saturated sodium chloride, twice with 1 N HCl, three times with saturated sodium chloride and water, in that order. The solution was dried with anhydrous sodium sulfate and concentrated in vacuo. Ether-hexane was added to the residue and the thus-precipitated material was filtered. Recrystallization was effected from ethyl acetate-ether-hexane to obtain the substance [13] (100.77 g, yield: 95.1%).
  • ethyl acetate 300 ml was added and 1 N HCl (60 ml) was added dropwise to adjust the pH of the aqueous layer to pH 2.
  • the aqueous layer was extracted with ethyl acetate (1 l. and 500 ml), and washed twice with saturated sodium chloride solution and water. After drying with anhydrous sodium sulfate, the ethyl acetate layer was concentrated in vacuo to obtain an oily material which was solidified by adding ether. Recrystallization was effected from ethyl acetate-ether to obtain the substance [15] (95.12 g, yield: 100%).
  • WSCI (8.88 ml, 48.52 mM) was added dropwise at -10° C. thereto and the mixture was stirred at room temperature for 2 days.
  • the DMF was distilled off in vacuo, and the residue was poured into ice cold water (2 l.).
  • the precipitate was filtered, suspended in water, washed and filtered. This operation was repeated five times at pH 6, and the product was dried in a desiccator in vacuo to obtain a solid (109 g) which was dissolved in methanol, concentrated in vacuo and azeotropically distilled by adding benzene to remove remaining water. This operation was repeated three times.
  • Recrystallization was effected from ethyl acetate-ether to obtain a material (90.09 g) which was dissolved in chloroform (270 ml) and charged on a column of silica gel (600 g) packed with chloroform, then eluted with chloroform-methanol (20:1). The fractions showing one spot upon TLC were collected and dried in vacuo to obtain the substance [15]. The remaining fractions were concentrated in vacuo and charged on a column of silica gel (220 g) and eluted the same way as above. The fractions showing one spot upon TLC were collected and dried in vacuo. The chromatographic operation above was repeated four times to obtain the substance [16] (total mount: 64.26 g, yield: 78.9%).
  • WSCI 28.6 ml, 156 mM
  • the DMF was distilled off and ethyl acetate (500 ml) was added to the residue, which was then washed with 5% sodium bicarbonate (three times), 1 N HCl (three times) and water (three times), in that order. After drying the ethyl acetate layer with anhydrous sodium sulfate, the organic layer was concentrated in vacuo.
  • the DMF was distilled off in vacuo and ethyl ether (400 ml) was added to the residue. After washing three times with 5% sodium bicarbonate solution, three times with 1 N HCl and three times with water, in that order, and drying with anhydrous magnesium sulfate, the solution was concentrated in vacuo. The thus-obtained oily material was dissolved in a small amount of benzene and charged on a column of silica gel (500 g) packed with benzene.
  • the residue was dissolved in ethyl acetate (500 ml), washed three times with 5% sodium bicarbonate solution, three times with 1 N HCl and three times with water, in that order, dried with anhydrous magnesium sulfate and concentrated in vacuo.
  • the residue was dissolved in a small amount of benzene and charged on a column of silica gel (250 g) packed with benzene.
  • BOC-Lys(Z-Cl)-OH prepared from BOC-Lys(Z-Cl)-OH.TBA (80 g, 163 mM) and HOBT (220 g, 163 mM) were added thereto; WSCI (29.8 ml, 163 mM) was added dropwise, and the mixture was stirred at 0° C. for one hour and at room temperature overnight.
  • the DMF was removed in vacuo, and ethyl acetate (600 ml) was added to the residue, which was then washed with 5% sodium bicarbonate solution, 1 N HCl and water, each three times, in that order.
  • reaction mixture was concentrated in vacuo, and ethyl acetate (1 l.) was added to the residue, which was then washed with 5% sodium bicarbonate solution, 1 N HCl and water, each three times, in that order. Hexane was added to the oily product, which was then crystallized by adding crystalline seed. The material was then recrystallized from ether-hexane to obtain the substance [28] (340.4 g, yield: 87.5%).
  • the substance [30] (98.4 g, 117 mM) was dissolved in DMF (300 ml). Hydrazine hydrate (100%) (117 ml) was added dropwise thereto and the mixture was stirred at room temperature for three hours. The DMF was distilled off in vacuo and ice water was added to the residue. The precipitate thus formed was filtered and dried. Recrystallization was effected from acetone to obtain the substance [31] (97.44 g, yield: 99%).
  • the substance [30] (60.6 g, 72 mM) was dissolved in DMF (300 ml) and 4.32 N HCl/dioxane solution (50 ml, 216 mM) was added dropwise at -50° C. After adding dropwise isoamylnitrile (11.5 ml, 80 mM) thereto, the mixture was stirred at -20° C. for 30 minutes. TEA (30 ml, 216 mM) was added at -50° C. to prepare an azide solution.
  • the substance [27] (53 g, 60 mM) was added to TEA (180 ml) at 0° C., and the mixture was stirred at room temperature for 30 minutes and the TEA was removed in vacuo.
  • Ether was added to the residue, and the formed precipitate was filtered, dissolved in DMF (100 ml) and neutralized with NMM (15 ml) at 0° C. This neutralized solution was added to the above azide solution and stirred at 0° C. overnight.
  • the solvent was distilled off in vacuo and ice water was added to the residue.
  • the precipitate was collected by filtration and washed with 1 N HCl, 5% sodium bicarbonate and water. Recrystallization was effected from methanol-DMF after drying in vacuo, to obtain the substance [32], (Yield: 95.9%)
  • the eluate and the washing solution were combined and lyophilized to obtain the product (1.46 g), which was dissolved in 8 M urea solution (30 ml), adjusted to pH 9 by adding aqueous ammonia at 0° C., then charged on a column (4.5 ⁇ 120 cm) of Sephadex LH-20 and eluted with 0.1 N acetic acid.
  • the eluate was fractionated into fractions of 7 ml each, and the fractions Nos. 55-83 were collected and then lyophilized to obtain a powder (990 mg).
  • the powder was dissolved in 0.1 N acetic acid (30 ml) and charged on a column (5 ⁇ 14 cm) of CMC.
  • H-Asp(OBzl)-OH 33.48 g, 150 mM was suspended in DMF (400 ml).
  • HOBT (2.03 g, 15 mM)
  • BOC-Gln-ONP 55.10 g, 150 mM
  • DMF 100 ml
  • the reaction mixture was adjusted to pH 7 by adding NMM (2 ml) at -10° C. and the mixture was stirred at room temperature overnight.
  • the pH of the mixture was adjusted three times to pH 7 by adding NMM (16 ml).
  • the DMF was removed in vacuo.
  • a citric acid solution 300 ml) and chloroform (300 ml) were added thereto and the mixture was shaken.
  • the aqueous layer was extracted with chloroform (300 ml and 100 ml). The chloroform layers were combined and washed four times with saturated sodium chloride, acidic water (pH 3) and acidic water (pH 4), in that order.
  • the aqueous layer was dried with anhydrous sodium sulfate and concentrated in vacuo. Ether was added to the residue and the precipitate formed was collected by filtration. Methanol was added to the collected precipitate and then the material was concentrated in vacuo, and then twice recrystallized from chloroform-ether to yield the substance [35] (32.31 g, yield: 47.7%).
  • the mother liquor was concentrated in vacuo and recrystallized twice from chloroform-ether to obtain the substance [35] (6.3 g).
  • Example 5 The substance [34] in Example 5 (101.10 g, 24 mM) was dissolved in methylene chloride (150 ml). TFA (350 ml) was added thereto at 0° C., and the mixture was stirred at room temperature for one hour. The TFA was distilled off in vacuo, ether was added to the residue, and the precipitate thus formed was filtered and dried over potassium hydroxide overnight (114.14 g).
  • the powder thus obtained was dissolved in DMF (300 ml) (pH 3), adjusted to pH 6 by adding NMM (8.64 ml) at 0° C., and substance [35] (13.00 g, 28.8 mM) and HOBT (3.89 g, 28.8 mM) were added to prepare a solution.
  • PCP (7.67 g, 28.8 mM) was added thereto, along with THF (50 ml) and more DMF (50 ml).
  • NMM (3.17 ml) was added to the solution at 0° C. (pH 5), and WSCI (5.27 ml) (pH 6) was added dropwise and the mixture was stirred at room temperature overnight.
  • AOC-Arg(Tos)-OH (63.82 g, 150 mM) was dissolved in DMF (200 ml).
  • H-Phe-OEt.HCl 36.18 g, 157.5 mM
  • HOBT 21.28 g, 157.5 mM
  • WSCI 28.82 ml, 157.5 mM
  • the DMF was removed in vacuo and a 5% sodium bicarbonate solution (500 ml) was added to the residue.
  • the precipitate thus formed was extracted twice with ethyl acetate (500 ml-300 ml).
  • WSCI 25.25 ml
  • WSCI 25.25 ml
  • the solvent was removed in vacuo and the residue was added to 5% sodium bicarbonate (600 ml), which was then extracted twice with ethyl acetate (300 ml).
  • the ethyl acetate layer was washed twice with 5% sodium bicarbonate, saturated sodium chloride solution, twice with 1 N HCl, three times with saturated sodium chloride solution and water, in that order, dried by adding anhydrous sodium sulfate, then concentrated in vacuo.
  • Amino acid analysis [0.529 mg/6 N HCl 0.5 ml, 105° C., 24 hours]: Asp 2.63 (3), Thr 0.97 (1), Ser 6.75 (8), Gln 1.92 (2), Pro 10.65 (10), Gly 1.03 (1), Ala 1.08 (1), Ile 0.93 (1), Leu 3 (3), Phe 0.77 (1), Lys 1.09 (1), Arg 1.72 (2).
  • the aqueous layer was adjusted to pH 3 by adding 1 N HCl (200 ml) to precipitate an oily material, which was extracted three times with ethyl acetate (300 ml), and the ethyl acetate layer was washed three times with aqueous sodium chloride and water. After drying with anhydrous sodium sulfate, the solution was concentrated in vacuo. Ether and hexane were added to the residue to obtain a precipitate. Recrystallization was effected with ethyl acetate-ether to yield the substance [42] (61.98 g, yield: 62.9%).
  • the resulting powder was dissolved in DMF (50 ml) (pH 3), adjusted to pH 7 by adding NMM (1.32 ml) at -10° C., the HOBT (0.53 g, 3.9 mM), a DMF solution (20 ml) of substance [42] (1.94 g, 3.9 mM), and a THF solution (20 ml) of PCP (1.04 g, 3.9 mM) were added thereto, in that order.
  • WSCI (0.71 ml, 3.9 mM) was added dropwise at -10° C. and stirred at -10° C. for two hours and at room temperature for three hours. More WSCI (0.71 ml) was added at -10° C.
  • This powder was dissolved in 8 M urea solution (350 ml), adjusted to pH 9.5 by adding aqueous ammonia, and charged on a column (4.5 ⁇ 14 cm) of CMC. 0.01 M ammonium acetate buffer (pH 4.5, 500 ml) was passed therethrough, and the column was then eluted with a linear gradient of 0.01 M (1.5 l.) to 0.2 M (1.5 l.) ammonium acetate buffer (pH 4.5). Fractions Nos. 48-59 (each fraction was 11.8 ml) were collected and lyophilized to yield a powder (800 mg).
  • Free mercapto groups 53.15% were detected according to the method "Colorimetric assay of cysteine by Ellman method".
  • H-Thr(Bzl)-OBzl.(COOH) 2 (68.88 g, 0.2 M) was dissolved in DMF (300 ml), the solution was neutralized by adding TEA (28 ml, 0.2 M), and HOBT (27.0 g, 0.2 M) and BOC-Leu-OH.H 2 O (49.86 g, 0.2 M) were added thereto.
  • WSCI (36.6 ml, 0.2 M) was added dropwise at 0° C. and the mixture was stirred at room temperature overnight. Then more WSCI (15 ml) was added and the mixture was again stirred overnight.
  • the DMF was distilled off in vacuo and the residue was dissolved in ethyl acetate (1.2 l.).
  • the ethyl acetate layer was washed with 5% sodium bicarbonate, water, 1 N HCl and water, in that order, dried by adding anhydrous sodium sulfate and concentrated in vacuo.
  • the oily substance thus obtained (99.24 g, yield: 94.2%) was allowed to stand for a long time to crystallize so as to obtain the substance [44].
  • the DMF was removed in vacuo, and the residue was dissolved in ethyl acetate (900 ml).
  • the ethyl acetate solution was washed with 5% sodium bicarbonate and water, and dried with anhydrous sodium sulfate, then concentrated in vacuo to obtain an oily material.
  • This oily material was dissolved in DMF (900 ml), HOBT (90 g) was added, and the mixture was stirred at room temperature for two days.
  • the DMF was distilled off in vacuo and the residue was dissolved in ethyl acetate (900 ml).
  • the solution was washed with 5% sodium bicarbonate and water, dried with anhydrous sodium sulfate, and then concentrated in vacuo.
  • TFA 180 ml
  • anisole 3 ml
  • the TFA was removed in vacuo and ether was added to the residue.
  • the precipitate was separated and dissolved in DMF (300 ml) and adjusted to pH 7 by adding TEA (26.04 ml, 187.29 mM) to obtain a de-BOC solution.
  • the substance [46] (52.44 g, 78.03 mM) was dissolved in DMF (300 ml). A 4.32 N HCl/dioxane solution (54.18 ml, 234.09 mM) was added thereto, then was added isoamylnitrile (11.55 ml, 85.83 mM), and then the mixture was stirred at -20° C. for 25 min. After adding TEA (32.52 ml, 234.09 mM) at -60° C., the above de-BOC solution was added thereto, and the mixture was stirred at 10° C. for three days. The DMF was removed in vacuo and the residue was dissolved in chloroform (900 ml).
  • the resulting powder was dissolved in DMF (60 ml), adjusted to pH 7 by adding NMM (1.98 ml) at -10° C., then HOBT (0.53 g, 3.9 mM), the substance [48] (4.84 g, 3.9 mM) and DMF (20 ml) were added thereto.
  • This powder was dissolved in 8 M urea solution (250 ml), adjusted to pH 9.5 by adding aqueous ammonia and poured on a column (4.5 ⁇ 17.5) of CMC. The column was washed with 0.01 M ammonium acetate buffer (pH 4.5) and gradiently eluted with 0.01 M (1.5 l) to 0.1 M (1.5 l.) ammonium acetate buffer (pH 4.5). Fractions Nos. 63-73 (each fraction was 12.4 ml) were collected and lyophilized to obtain another powder material (930 mg).
  • Example 6 The substance [41] (8.44 g, 1.5 mM) in Example 6 was dissolved in DMF (35 ml) (pH 3) and the solution was adjusted to pH 7 by adding NMM (0.48 ml) at -10° C.
  • WSCI (0.35 ml, 1.95 mM) was added dropwise at -10° C. and the mixture was stirred at -10° C. for two hours and at room temperature for three hours. More WSCI (0.36 ml, 1.95 mM) was added dropwise in the reaction mixture at -10° C. Still more WSCI (0.11 ml, 0.6 mM) was added dropwise at -10° C. and the mixture was stirred at room temperature overnight. The DMF was removed in vacuo and the residue was added to cold water (400 ml). The precipitate was suspended and washed three times and dried in vacuo for two days. The dried material was dissolved in methanol-chloroform (1:2) and reprecipitated by adding ether, and this was repeated six times to obtain the substance [56]. (11.59 g, yield: 109.9%).
  • This powder was dissolved in 10% mercapto ethanol in an 8 M urea solution (120 ml), adjusted to pH 9.5 by adding aqueous ammonia and the superjacent atmosphere was replaced by nitrogen gas. The mixture was then stirred for 30 minutes. The solution was poured on a column (4.5 ⁇ 12 cm) of CMC, which was washed with 0.1 M ammonium acetate buffer (pH 4.5). Elution was carried out by linear gradient elution of 0.01 M (1 l.) to 0.1 M (1 l.) ammonium acetate (pH 4.5). Fractions Nos.
  • Substance [41] (8.44 g, 1.5 mM) in Example 6 was dissolved in DMF (35 ml) (pH 3) and adjusted to pH 7 by adding NMM (0.48 ml) at -10° C.
  • HOBT (0.26 g, 1.95 mM)
  • BOC-Tyr(Bzl-Cl 2 )-Gly-Gly-Pro-Lys(Z)-Asp(OBzl)-His-Pro-Ley-Thr(Bzl)-Cys(Acm)-Asp(OBzl)-OH (3.97 g, 1.95 mM) were added, then a THF solution (5 ml) of PCP (0.52 g, 1.95 mM) was added thereto.
  • WSCI (0.36 ml, 1.95 mM) was added dropwise at -10° C., and the mixture was stirred at -10° C. for two hours and at room temperature for three hours. More WSCI (0.36 ml, 1.95 mM) was added dropwise at -10° C. to the reaction mixture, which was stirred at room temperature overnight. Still more WSCI (0.11 ml, 0.6 mM) was added dropwise thereto at -10° C. and the mixture was stirred at room temperature overnight. The DMF was removed in vacuo, and the residue was poured into cold water (400 ml). The operations of suspending the precipitate in water and filtering were repeated three times, and then the material was dried in vacuo for two days. This dried material was dissolved in chloroform-methanol (2:1) and reprecipitated with ether. This operation was repeated six times to obtain the substance [60] (12.03 g).
  • the combined powder (4.80 g) was dissolved in 8 M urea solution (250 ml), adjusted to pH 9 by adding aqueous ammonia, and was poured on a column (3.5 ⁇ 17 cm) of CMC.
  • the column was washed with 0.01 M ammonium acetate buffer (pH 4.5, 500 ml) and linear gradient elution was performed with 0.01 M (1.2 l) to 0.1 M (1.2 l.) ammonium acetate buffer (pH 4.5).
  • Fractions Nos. 52-60 each fraction was 12 ml) were collected and lyophilized to obtain a powder (1.72 g).
  • Amino acid analysis (under conditions of protected peptide decomposition): Asp (4.96 (5), Thr 1.15 (2), Ser 6.26 (8), Gln 2.04 (2), Pro 12.14 (12), Gly 2.93 (3), Ala 0.93 (1), Cys-Cys 0.45 (0.5), Ile 0.93 (1), Leu 4 (4), Tyr 0.99 (1), Phe 1.00 (1), Lys 1.98 (2), His 0.97 (1), Arg 2.05 (2).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US06/320,699 1980-11-11 1981-11-12 C-Terminal fragment of human chorionic gonadotropin Expired - Fee Related US4400316A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP55158567A JPS5781447A (en) 1980-11-11 1980-11-11 Human chorionic gonadotropic hormone c-terminal fragment
JP55-158567 1980-11-11

Publications (1)

Publication Number Publication Date
US4400316A true US4400316A (en) 1983-08-23

Family

ID=15674511

Family Applications (1)

Application Number Title Priority Date Filing Date
US06/320,699 Expired - Fee Related US4400316A (en) 1980-11-11 1981-11-12 C-Terminal fragment of human chorionic gonadotropin

Country Status (4)

Country Link
US (1) US4400316A (de)
JP (1) JPS5781447A (de)
DE (1) DE3144818A1 (de)
GB (1) GB2091740B (de)

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4517290A (en) * 1980-03-31 1985-05-14 Takeda Chemical Industries, Ltd. Method for enzyme immunoassay and peptide-enzyme conjugate and antibody therefor
US4691006A (en) * 1983-03-04 1987-09-01 Ohio State University Antigenic modification of polypeptides
US4713366A (en) * 1985-12-04 1987-12-15 The Ohio State University Research Foundation Antigenic modification of polypeptides
US4762913A (en) * 1973-05-07 1988-08-09 The Ohio State University Antigenic modification of polypeptides
US4767842A (en) * 1973-05-07 1988-08-30 The Ohio State University Antigenic modification of polypeptides
US4855285A (en) * 1985-12-04 1989-08-08 The Ohio State University Research Foundation Antigenic modification of polypeptides
US5338835A (en) * 1989-02-21 1994-08-16 Washington University CTP-extended form of FSH
US5380668A (en) * 1993-07-06 1995-01-10 University Of Utah Research Foundation Compounds having the antigenicity of hCG
WO1997049373A2 (en) * 1996-06-24 1997-12-31 University Of Maryland Biotechnology Institute Treatment and prevention of hiv infection by administration of derivatives of human chorionic gonadotropin
US5712122A (en) * 1989-02-21 1998-01-27 Washington University Carboxy terminal peptide-extended proteins
US5759818A (en) * 1991-10-04 1998-06-02 Washington University N-terminal CTP extended pharmaceutical peptides and proteins
US5817753A (en) * 1985-12-04 1998-10-06 The Ohio State University Research Foundation Antigenic modification of polypeptides
US5968513A (en) * 1996-06-24 1999-10-19 University Of Maryland Biotechnology Institute Method of promoting hematopoiesis using derivatives of human chorionic gonadotropin
US5997871A (en) * 1996-06-24 1999-12-07 University Of Maryland Biotechnology Insitute Treatment and prevention of cancer by administration of derivatives of human chorionic gonadotropin
US6039948A (en) * 1973-05-07 2000-03-21 The Ohio State University Method for treatment of antigenically modified polypeptides
US6096318A (en) * 1973-05-07 2000-08-01 The Ohio State University Antigenically modified HCG polypeptides
US6143305A (en) * 1973-05-07 2000-11-07 The Ohio State University Antigenically modified polypeptides composition
US6583109B1 (en) 1997-06-24 2003-06-24 Robert C. Gallo Therapeutic polypeptides from β-hCG and derivatives
US20060205672A1 (en) * 2000-07-21 2006-09-14 Schering Corporation Novel peptides as NS3-serine protease inhibitors of hepatitis C virus
US20100081614A1 (en) * 2006-02-03 2010-04-01 Fuad Fares Long-acting growth hormone and methods of producing same
US20100317585A1 (en) * 2006-02-03 2010-12-16 Udi Eyal Fima Long-acting coagulation factors and methods of producing same
US7994278B1 (en) 1999-08-06 2011-08-09 Nobel Biosciences Llc Biologically active polypeptides derived from a novel early stage pregnancy factor designated maternin (MA)
US8323636B2 (en) 2006-02-03 2012-12-04 Prolor Biotech Ltd. Long-acting interferons and derivatives thereof and methods thereof
US8426166B2 (en) 2006-02-03 2013-04-23 Prolor Biotech Inc. Long-acting polypeptides and methods of producing same
US8450269B2 (en) 2006-02-03 2013-05-28 Prolor Biotech Ltd. Long-acting growth hormone and methods of producing same
US8465948B2 (en) 2006-02-03 2013-06-18 Prolor Biotech Ltd. Long-acting veterinary polypeptides and methods of producing and administering same
US8530419B2 (en) 2003-10-03 2013-09-10 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US8759292B2 (en) 2006-02-03 2014-06-24 Prolor Biotech, Llc Long-acting coagulation factors and methods of producing same
US8905913B2 (en) 2009-04-23 2014-12-09 Jbs United Animal Health Ii Llc Method and composition for synchronizing time of insemination
US8946155B2 (en) 2006-02-03 2015-02-03 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9249407B2 (en) 2006-02-03 2016-02-02 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9458444B2 (en) 2006-02-03 2016-10-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9522945B2 (en) 2012-04-19 2016-12-20 Opko Biologics Ltd. Long-acting oxyntomodulin variants and methods of producing same
US9663778B2 (en) 2009-07-09 2017-05-30 OPKO Biologies Ltd. Long-acting coagulation factors and methods of producing same
US9724380B2 (en) 2012-11-28 2017-08-08 Jbs United Animal Health Ii Llc Method and compositions for synchronizing time of insemination in gilts
US9808534B2 (en) 2012-11-20 2017-11-07 Opko Biologics Ltd. Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
US9828417B2 (en) 2006-02-03 2017-11-28 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9884901B2 (en) 2006-02-03 2018-02-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10221228B2 (en) 2006-02-03 2019-03-05 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10351615B2 (en) 2006-02-03 2019-07-16 Opko Biologics Ltd. Methods of treatment with long-acting growth hormone
US10960058B2 (en) 2015-06-19 2021-03-30 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US11197915B2 (en) 2013-10-21 2021-12-14 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US11976106B2 (en) 2016-07-11 2024-05-07 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1186733B (it) * 1985-06-05 1987-12-16 Eniricerche Spa Composti tripeptidici ad azione ipotensiva
ATE143669T1 (de) * 1988-07-20 1996-10-15 Abbott Lab Hcg-peptide zur verwendung in antikörper- reinigungsverfahren
IT1237474B (it) * 1989-10-05 1993-06-07 Polifarma Spa Composti tripeptidici e loro uso farmaceutico come immuno-modulatori
US6737515B2 (en) 1993-11-19 2004-05-18 Washington University Follicle stimulating hormone-glycosylation analogs

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS56138248A (en) * 1980-03-31 1981-10-28 Takeda Chem Ind Ltd Enzyme immunity measuring method of artificial woolen gonadotropine
JPS576363A (en) * 1980-06-13 1982-01-13 Takeda Chem Ind Ltd Production of specific antibody

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Kawaski et al., Chem. Pharm. Bull. 28(9) 2692-2698, 2699-2706 (1980). *

Cited By (77)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4762913A (en) * 1973-05-07 1988-08-09 The Ohio State University Antigenic modification of polypeptides
US4767842A (en) * 1973-05-07 1988-08-30 The Ohio State University Antigenic modification of polypeptides
US6143305A (en) * 1973-05-07 2000-11-07 The Ohio State University Antigenically modified polypeptides composition
US6096318A (en) * 1973-05-07 2000-08-01 The Ohio State University Antigenically modified HCG polypeptides
US6039948A (en) * 1973-05-07 2000-03-21 The Ohio State University Method for treatment of antigenically modified polypeptides
US4517290A (en) * 1980-03-31 1985-05-14 Takeda Chemical Industries, Ltd. Method for enzyme immunoassay and peptide-enzyme conjugate and antibody therefor
US4691006A (en) * 1983-03-04 1987-09-01 Ohio State University Antigenic modification of polypeptides
US5817753A (en) * 1985-12-04 1998-10-06 The Ohio State University Research Foundation Antigenic modification of polypeptides
US4713366A (en) * 1985-12-04 1987-12-15 The Ohio State University Research Foundation Antigenic modification of polypeptides
US4855285A (en) * 1985-12-04 1989-08-08 The Ohio State University Research Foundation Antigenic modification of polypeptides
US6217881B1 (en) 1985-12-04 2001-04-17 The Ohio State University Research Foundation Antigenic modification of HCG polypeptides
US5891992A (en) * 1985-12-04 1999-04-06 The Ohio State University Research Foundation Antigenic modification of polypeptides
US5712122A (en) * 1989-02-21 1998-01-27 Washington University Carboxy terminal peptide-extended proteins
US5338835A (en) * 1989-02-21 1994-08-16 Washington University CTP-extended form of FSH
US5759818A (en) * 1991-10-04 1998-06-02 Washington University N-terminal CTP extended pharmaceutical peptides and proteins
US5380668A (en) * 1993-07-06 1995-01-10 University Of Utah Research Foundation Compounds having the antigenicity of hCG
US6699656B2 (en) 1996-06-24 2004-03-02 University Of Maryland Biotechnology Institute Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin
WO1997049373A2 (en) * 1996-06-24 1997-12-31 University Of Maryland Biotechnology Institute Treatment and prevention of hiv infection by administration of derivatives of human chorionic gonadotropin
WO1997049373A3 (en) * 1996-06-24 1998-02-26 Univ Maryland Biotech Inst Treatment and prevention of hiv infection by administration of derivatives of human chorionic gonadotropin
US6319504B1 (en) 1996-06-24 2001-11-20 University Of Maryland Biotechnology Institute Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin
US5997871A (en) * 1996-06-24 1999-12-07 University Of Maryland Biotechnology Insitute Treatment and prevention of cancer by administration of derivatives of human chorionic gonadotropin
US5968513A (en) * 1996-06-24 1999-10-19 University Of Maryland Biotechnology Institute Method of promoting hematopoiesis using derivatives of human chorionic gonadotropin
US6583109B1 (en) 1997-06-24 2003-06-24 Robert C. Gallo Therapeutic polypeptides from β-hCG and derivatives
US6596688B1 (en) 1997-06-24 2003-07-22 University Of Maryland Biotechnology Institute Method for promoting hematopoiesis
US6620416B1 (en) 1997-06-24 2003-09-16 University Of Maryland Biotechnology Institute Method for treating HIV
US6699834B1 (en) 1997-06-24 2004-03-02 University Of Maryland Biotechnology Institute Method for treating cancer
US6805882B1 (en) 1997-06-24 2004-10-19 University Of Maryland Biotechnology Institute Therapeutic fractions of sources of HCG
US7994278B1 (en) 1999-08-06 2011-08-09 Nobel Biosciences Llc Biologically active polypeptides derived from a novel early stage pregnancy factor designated maternin (MA)
US9175077B2 (en) 1999-08-06 2015-11-03 Nobel Biosciences Llc Nucleic acids encoding biologically active polypeptides derived from a novel early stage pregnancy factor designated maternin (MA)
US20060205672A1 (en) * 2000-07-21 2006-09-14 Schering Corporation Novel peptides as NS3-serine protease inhibitors of hepatitis C virus
US20110117057A1 (en) * 2000-07-21 2011-05-19 Schering Corporation Novel peptides as ns3-serine protease inhibitors of hepatitis c virus
USRE43298E1 (en) 2000-07-21 2012-04-03 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US10028996B2 (en) 2003-10-03 2018-07-24 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US9351818B2 (en) 2003-10-03 2016-05-31 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US10898539B2 (en) 2003-10-03 2021-01-26 Thorn BioSciences LLC Process for the synchronization of ovulation for timed breeding without heat detection
US9018165B2 (en) 2003-10-03 2015-04-28 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US8937044B2 (en) 2003-10-03 2015-01-20 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US8530419B2 (en) 2003-10-03 2013-09-10 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US8927496B2 (en) 2003-10-03 2015-01-06 Thorn Bioscience Llc Process for the synchronization of ovulation for timed breeding without heat detection
US8465948B2 (en) 2006-02-03 2013-06-18 Prolor Biotech Ltd. Long-acting veterinary polypeptides and methods of producing and administering same
US8304386B2 (en) 2006-02-03 2012-11-06 Prolor Biotech, Inc. Long-acting growth hormone and methods of producing same
US11066658B2 (en) 2006-02-03 2021-07-20 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US8476234B2 (en) 2006-02-03 2013-07-02 Prolor Biotech Inc. Long-acting coagulation factors and methods of producing same
US8450269B2 (en) 2006-02-03 2013-05-28 Prolor Biotech Ltd. Long-acting growth hormone and methods of producing same
US8946155B2 (en) 2006-02-03 2015-02-03 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US8999670B2 (en) 2006-02-03 2015-04-07 Opko Biologics Ltd. Long-acting polypeptides and methods of producing same
US8426166B2 (en) 2006-02-03 2013-04-23 Prolor Biotech Inc. Long-acting polypeptides and methods of producing same
US8323636B2 (en) 2006-02-03 2012-12-04 Prolor Biotech Ltd. Long-acting interferons and derivatives thereof and methods thereof
US9249407B2 (en) 2006-02-03 2016-02-02 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US8759292B2 (en) 2006-02-03 2014-06-24 Prolor Biotech, Llc Long-acting coagulation factors and methods of producing same
US10119132B2 (en) 2006-02-03 2018-11-06 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9458444B2 (en) 2006-02-03 2016-10-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US20100081614A1 (en) * 2006-02-03 2010-04-01 Fuad Fares Long-acting growth hormone and methods of producing same
US10640758B2 (en) 2006-02-03 2020-05-05 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US10351615B2 (en) 2006-02-03 2019-07-16 Opko Biologics Ltd. Methods of treatment with long-acting growth hormone
US10221228B2 (en) 2006-02-03 2019-03-05 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10144921B2 (en) 2006-02-03 2018-12-04 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9828417B2 (en) 2006-02-03 2017-11-28 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9884901B2 (en) 2006-02-03 2018-02-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US9896494B2 (en) 2006-02-03 2018-02-20 Opko Biologics Ltd. Long-acting polypeptides and methods of producing same
US9908924B2 (en) 2006-02-03 2018-03-06 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US20100317585A1 (en) * 2006-02-03 2010-12-16 Udi Eyal Fima Long-acting coagulation factors and methods of producing same
US10030060B2 (en) 2006-02-03 2018-07-24 Opko Biologics Ltd. Long-acting polypeptides and methods of producing same
US9352011B2 (en) 2009-04-23 2016-05-31 Jbs United Animal Health Ii Llc Method and composition for synchronizing time of insemination
US8905913B2 (en) 2009-04-23 2014-12-09 Jbs United Animal Health Ii Llc Method and composition for synchronizing time of insemination
US9757425B2 (en) 2009-04-23 2017-09-12 Jbs United Animal Health Ii Llc Method and composition for synchronizing time of insemination
US10668127B2 (en) 2009-04-23 2020-06-02 United-Ah Ii, Llc Method and composition for synchronizing time of insemination
US10538755B2 (en) 2009-07-09 2020-01-21 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US9663778B2 (en) 2009-07-09 2017-05-30 OPKO Biologies Ltd. Long-acting coagulation factors and methods of producing same
US9522945B2 (en) 2012-04-19 2016-12-20 Opko Biologics Ltd. Long-acting oxyntomodulin variants and methods of producing same
US9808534B2 (en) 2012-11-20 2017-11-07 Opko Biologics Ltd. Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
US10376558B2 (en) 2012-11-28 2019-08-13 United-Ah Ii, Llc Method and compositions for synchronizing time of insemination in gilts
US9724380B2 (en) 2012-11-28 2017-08-08 Jbs United Animal Health Ii Llc Method and compositions for synchronizing time of insemination in gilts
US11197915B2 (en) 2013-10-21 2021-12-14 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US12029780B2 (en) 2013-10-21 2024-07-09 Opko Biologics Ltd. Long-acting polypeptides and methods of producing and administering same
US10960058B2 (en) 2015-06-19 2021-03-30 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same
US11976106B2 (en) 2016-07-11 2024-05-07 Opko Biologics Ltd. Long-acting coagulation factors and methods of producing same

Also Published As

Publication number Publication date
JPS5781447A (en) 1982-05-21
GB2091740A (en) 1982-08-04
GB2091740B (en) 1984-03-28
DE3144818A1 (de) 1982-08-19

Similar Documents

Publication Publication Date Title
US4400316A (en) C-Terminal fragment of human chorionic gonadotropin
US4086221A (en) Polypeptides and process for producing the same
Schwabe et al. Primary structure of the B-chain of porcine relaxin
US4656250A (en) [Nle8, Nle18, Tyr34 or Phe34 ]-h-PTH peptide derivatives
US4529595A (en) GRF Analogs
US4607023A (en) Natriuretic
US4423034A (en) Process for the preparation of antibodies
JPH0689035B2 (ja) Ｇｒｆ類似体類
US4271068A (en) Process for the manufacture of cystine-containing peptides
US4610976A (en) Porcine GRF
DK162649B (da) Analogifremgangsmaade til fremstilling af pgrf eller pgrf-analoge peptider, farmaceutisk acceptable additionssalte heraf samt mellemprodukt til brug ved fremgangsmaaden
CA1247604A (en) Ovine growth hormone releasing factor
EP0352014A2 (de) Zyklische GRF-Analoge
EP0173384B1 (de) Polypeptid verwandt mit alpha-hANP
EP0137689B1 (de) GRF-Analoge
US4652627A (en) Calcitonin analogs with C-terminal D-amino acid substituents
CA1094051A (en) Polypeptide having serum calcuim reducing activity
US4703106A (en) Novel polypeptide and process for producing the same
CA1247599A (en) Mammalian pgrf
US4703035A (en) Human pancreatic GRF amidated fragments
US4118380A (en) Decapeptide analogs of somatostatin
JP2685195B2 (ja) Ｇｒｆ類縁体ｖ
EP0298474B1 (de) Calcitonin-Derivate und deren Salze
Boissonnas et al. Chemistry of the neurohypophysial hormones
US4416820A (en) Indole derivatives and a method for production of peptides

Legal Events

Date Code Title Description
AS Assignment

Owner name: TOYO JOZO KABUSHIKI KAISHA, 632-1, MIFUKU, OHITO-C

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:KATSURAGI, SHIGEO;MORITA, KAORU;KOBARI, SADAMI;AND OTHERS;REEL/FRAME:003955/0287

Effective date: 19811103

Owner name: TOYO JOZO KABUSHIKI KAISHA, A CORP. OF JAPAN, JAPA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KATSURAGI, SHIGEO;MORITA, KAORU;KOBARI, SADAMI;AND OTHERS;REEL/FRAME:003955/0287

Effective date: 19811103

FEPP Fee payment procedure

Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

DI Adverse decision in interference

Effective date: 19870212

LAPS Lapse for failure to pay maintenance fees
STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 19870823